Biological activity in blood, such as bacteremia (bacteria in blood), remains a significant problem despite the availability of antimicrobial drugs. In particular, the rapid isolation of offending organisms from bacteremic patients is made more difficult when the patient has been administered antibiotics, which are transferred along with the bacteria in the blood into culture broths and often inhibit the growth of the organism. It is nevertheless important that the identification and susceptibility of an infecting organism to various antimicrobial drugs be determined as early as possible in the course of bacteremia.
When conventional culturing techniques are employed to identify an infecting organism, the administration of an antibiotic prior to drawing the blood sample for testing can result in inhibition of the growth of bacteria, thus interfering with isolation and identification of the bacteria. Even when antibiotics may not be present in the blood, the isolation of the offending organism(s) still may require excess periods of incubation because of inhibitors contained in serum, plasma, or lysed erythrocytes.
Similar problems exist despite the type of body fluid being examined, whether it is urine, spinal fluid, abscess exudates, serum, pertioneal fluid and the like.
Antibiotics inhibit bacteria through a variety of mechanisms. One of these mechanisms is to inhibit the bacteria's ability to synthesize its cell wall. Inhibition of cell wall synthesis is caused by antibiotics belonging to the .beta.-lactam or cephalosporin groups. This includes such examples as penicillin G, amipicillin, amoxicillin, carbenicillin, nafcillin, ticarcillin, cefamandole, cefotaxime, cefoxitin, cephalexin, cephaloridine, cephalothin and moxalactam.
A primary function of the bacterial cell wall is to allow the cell to live in a fluid environment which is less dense than the interior of the cell. Because the bacterium's cytoplasm (interior) is much more dense than the fluid surrounding the cell, there is a large osmotic pressure difference. This causes water to be drawn into the bacterium and were it not for its rigid cell wall, the cell would swell and burst. When synthesis of the cell wall is inhibited by antibiotics, the cell wall is weakened and osmotic pressure can cause lysis (bursting) and death of the bacteria.
Weakening of the bacterium's cell wall by an antibiotic does not take place instantaneously, but usually over a period of hours. The time required is affected by several factors including the concentration of antibiotics and the rate of growth of the bacterium. If a patient has a bacterial infection of the blood and is being treated with a -lactam or cephalosporin class antibiotic, there is a probability that at a given time during treatment there is a population of bacteria in the blood which has been weakened, but not killed by the antibiotic. A sample of this patient's blood when added to normal blood culture medium may show no viable bacteria to be present, for cell wall damaged bacteria in the blood may lyse due to osmotic stress in the culture medium. This would be a false negative result.
Adding a carbohydrate saccharide, such as sucrose (for example) to a culture medium increases its density and such media are described as hypertonic. In hypertonic media, there is less of a difference in density between the bacterium's cytoplasm and the surrounding fluid. This causes less of an osmotic differential and therefore less osmotic stress on the bacterial cell. Cell wall-damaged bacteria can thus survive more easily in hypertonic media.
The usual method for detection of bacteria is to inoculate 5 ml of a body fluid into a culture medium and wait for the appearance or turbidity which is an indication of bacterial growth. Patients who have been subjected to antibiotic therapy will have the antibiotic present in the body fluid at the time the fermentation is initiated. Presence of the antibiotic inhibits growth of the bacteria and may delay isolation of the bacteria for as long as 14 days.
More recently, a radiometric technique for the detection of biological activity in the blood has undergone clinical testing and has been adopted for commercial practice. In that method, samples of blood are inoculated into a suitable growth medium that includes a C.sup.14 containing carbon source, the inoculated medium is incubated for a suitable period, and a portion of the gaseous atmosphere is analyzed for C.sup.14 O.sub.2 while in the gaseous state. Such process is described, inter alia, in U.S. Pat. No. 3,676,679 issued July 11, 1972; and in the articles "Early Detection of Bacterial Growth, with Carbon.sup.14 Labeled Glucose," Radiology, 92, No. 1, pp. 154-5 (January 1969); "Automated Radiometric Detection of Bacterial Growth in Blood Culture," J. Labs. Clin. Med., 75, No. 3, pp. 529-34 (March 1970); and "Automated Radiometric Detection of Bacteria in 2,967 Blood Cultures," Applied Microbiology, 22, No. 5, pp. 846-849 (November 1979). A commercial instrument for the practice of a rapid, automated process is available under the trademark BACTEC (Johnston Laboratories). Although this method can be used to rapidly determine the presence of bacteria in a culture in the absence of antibiotics or their inhibitors, it is inefficient when growth is attempted in the presence of antibiotics or other inhibitors in the culture media.
Antibiotics can be separated from microorganisms by membrane chromatography, but these procedures are not practical because of the complexity of the separation technique and the high rate of contamination of the test culture.
U.S. Pat. No. 4,174,277 to Melnick et al. discloses a method for separation of antibiotics from microorganisms present in a body fluid sample. In the method of the Melnick patent, an antibiotic is selectively removed from a bacterially infected body fluid specimen by adsorbing the antibiotic onto a resin system treated with a detergent.
The detergent renders the resin system selective for the antibiotic while permitting the bacteria to remain free in the eluting fluid and thus the bacteria are separated from the antibiotic. The eluted body fluid specimen containing the bacteria is then inoculated into a growth medium and cultured.
While the method of the Melnick et al. patent has been useful in overcoming the problem of antibiotic inhibitor contamination of blood samples, it introduces a separate handling step in the treatment of body fluid samples prior to the conventional culturing process. The separate handling step is particularly cumbersome and may introduce contamination when handling a multitude of samples as is done with current automatic fermentation apparatus.
It is therefore, a principal object of the present invention to provide a culture medium for the growth and detection of an infecting organism in a body fluid specimen without the need to resort to a separate step for separation of an antibiotic or inhibiting substance.
It is another object of this invention to provide a means for isolating antimicrobial inhibitors from body fluid specimens during growth of an organism in culture medium.
It is a further object of the present invention to provide a means for isolating antibiotics from an infected body fluid specimen without significantly affecting the metabolism of the microorganism population of the specimen during growth in culture medium.
It is yet another object of the present invention to provide a method utilizing a resin which will isolate antibiotics and other inhibitors contained in a body fluid specimen while exhibiting little effect on growth of microorganisms in the specimen.
It is still another object of the present invention to provide a resin for isolating materials inhibitory to microorganisms in a body fluid specimen without affecting the microbial population of the specimen.